ABSTRACT Pulmonary vascular disease (PVD) is an important source of morbidity and mortality in patients with congenital heart disease (CHD). The natural history of PVD in these patients reveals the importance of abnormal pulmonary blood flow (PBF) and pressure in the pathophysiology. Patients with cardiac defects that expose the pulmonary vasculature to increased flow and direct pressure from the systemic ventricle develop PVD with a greater incidence and severity than patients with defects that result in increased PBF alone. Pulmonary endothelial cells (EC) are integral mediators of disease, due to their exposure to these normal and abnormal hemodynamic (mechanical) forces including shear stress, hydrostatic pressure, and cyclic strain. Our laboratory has developed two distinct, clinically relevant models of CHD in fetal lambs: (1) left pulmonary artery (LPA) ligation that results in increased PBF alone to the right lung; and (2) aortopulmonary shunt placement that results in increased PBF and a direct pressure stimulus. Our preliminary data demonstrate that at 4-6 weeks of age, model lambs manifest distinct aberrations in endothelial cell signaling and vascular function. For example, RNAseq analysis on primary pulmonary artery endothelial cells from each lamb model demonstrates markedly distinct gene expression patterns, and studies in isolated vessels demonstrate disparate alterations in vascular reactivity. Moreover, we have generated novel in vivo and in vitro data demonstrating that pressure- induced ET-1 production triggers a signaling cascade resulting in: endothelial dysfunction, disturbed mitochondrial bioenergetics, and a hyper-proliferative, anti-apoptotic endothelial cell phenotype. Our overall hypothesis is that the distinct mechanical forces associated with increased PBF versus increased PBF and direct pressure induce patterned alterations in gene expression and vascular function that underlie the incidence and progression of PVD associated with CHD. Specifically, we hypothesize that pressure (cyclic stretch)-induced ET-1 upregulation results in early endothelial dysfunction and a proliferation phenotype secondary to mitochondrial remodeling with ROS production and HIF-1? stabilization. We further hypothesize that flow-alone maintains endothelial function, but triggers shear-induced c-MYC activation and increased O2 consumption, resulting in a phenotype that is predisposed to proliferation. The current proposal is based on large animal, clinically relevant models of CHD that require fetal cardiac surgical techniques. This platform supports integrated whole animal, isolated vessel, biochemical, cellular, and molecular studies to elucidate the pathobiology of different abnormal mechanical forces within the pulmonary vasculature. More specifically, our investigations are focused on endothelial cell mechanosensor signal transduction, and thus have the potential to expose therapeutic targets that are more precisely linked to specific pathophysiologic states. This, in turn, would allow for the development of novel lesion-specific treatment strategies, which would represent a fundamental advance compared to currently available nonspecific treatment approaches.